The interaction of molecules on the surface of living cells is of great interest to biologists. In many cases, the interaction partners are mobile, so their common movement can be used as proxy information on their interaction. The size of intermolecular interaction regions is of the order of 10-100 nm. Hence, an analysis based on imaging the two molecules with a light microscope is of limited value, since the resolution limit of a light microscope is about 300 nm. Since the measurements are to be made on living cells, electron microscopes cannot be utilized. Optical super-resolution techniques can, in principle, provide the required resolution. However, the time required to form a high-resolution image with such techniques is too long to observe the co-localization or co-movement of typically mobile molecules. In particular, the molecules in question travel significant distances during the imaging process, and hence, the resolution is blurred by the molecular motion. It is inherently difficult to reduce this effect by lowering the imaging time, as the molecules show diffusion, where the expected displacement over time increases with the square root of the time. Hence, a four-fold reduction in imaging time will only result in a two-fold reduction of motion blur.
Foerster Resonant Energy Transfer (FRET) attempts to overcome these problems by labeling the molecules in question with two appropriately chosen different dyes. If the molecules in question approach one another within about 5 nm, a shift in the color of the florescence emission occurs. However, to detect the shift, there must be a significant relative number of molecules within 5 nm of each other. Since molecules do not usually approach one another within this distance for a prolonged time, the fraction of the molecules contributing to the shifted spectrum is often too small to be detected. In addition, molecules can still reliably interact, e.g. through a third intermediary partner, but mostly remain separated more than ˜10 nm, preventing FRET. An example where such a situation is expected to occur is within hypothesized structures called lipid rafts. Here, lipids in the cell membrane form islets which are enriched in certain molecule species. However, the molecules within the rafts are thought to be mobile, and are not expected to be packed tightly enough to result in observable FRET.
Fluorescence Correlation Spectroscopy (FCS) attempts to overcome the resolution problems by inferring that the molecules move together. Consider the case in which one wishes to determine if a first molecule moves with a second molecule. In FCS, the first molecules are tagged with a first label, and the second molecules are tagged with a second label having a different color. Only a small region of the cell's membrane is illuminated (a spot about 300 nm in diameter). The optical emission from the region is recorded at very high speeds. Since the region is large enough to accommodate many molecules of both species at the same time, there will always be a signal with both colors. That signal will vary over time depending on the molecular movements. If the molecules are mobile and travel together, the changes in signals among the two color channels will be correlated. If the two molecules do not travel together, the channels will be uncorrelated. In practice, the signals are very noisy and require statistical post processing to detect any co-localization.
Another proposed solution is referred to as Thinning Out Clusters While Conserving the Stoichiometry of Labeling (TOCCSL). Here, the target molecules are also labeled with fluorescent dyes. A small region of the cell's membrane is photo-bleached exhaustively using a laser beam to eliminate fluorescence from the target molecules in that region. Subsequently, diffusion of the molecules leads to a gradual exchange of molecules between the bleached region and its surroundings. After a time of typically less than a second, an image of the bleached region is taken with a light microscope. At that time, only a small number of bleached (now invisible) molecules will have been replaced by non-bleached (visible) molecules. Hence, the visible molecules will be, on average, separated by a distance of about 1 micron in the formerly bleached region. At this separation, the unbleached molecules that have moved into the bleached region will appear as individual fluorescent spots. By measuring the brightness of a spot and the number of dye molecules in the spot, the number of molecules of interest in that spot can be determined. If the molecules do not move together, each spot would be expected to have a brightness consistent with that of a single dye molecule. If the molecules move in clusters of N molecules, then the brightness of the spot would be N times greater. If two-color labeling is used for two different species of molecules, the color ratio of the spots provides information on the relative composition of the molecule clusters. Finally, if the time between bleaching and imaging is varied, the temporal stability of the clusters on a time frame of the order of milliseconds to seconds can be determined.
This technique assumes that groups of molecules are either fully bleached or unbleached. The main problem with the TOCCSL technique is that the border of the bleached region is slightly blurred due to the optical resolution limit of the bleaching light beam. In addition, some molecules move back and forth between the bleached region and non-bleached region during the bleaching process. As a result, the groups in this region will not necessarily be completely bleached. These partially bleached groups complicate the interpretation of the data. Accordingly, a method that reduces the number of partially-bleached groups is needed.